Isolation of Azospirillum lipoferum from the rhizosphere of rice by a new, simple method

A new, simple method for isolating Azospirillum strains from the roots and the rhizosphere of rice is described. The method is based on the capacity of Azospirillum, a nitrogen-fixing bacterial genus, to grow in nutrient-deficient liquid media such as distilled water, KCl (8.5 g/L), or soil extract medium. The enrichment efficiency of the deficient media was compared with classical N-free malate medium. Serial dilutions from 10(-1) to 10(-10) of rice root macerates and rhizosphere soil were incubated at 28 degrees C in these media without agitation for 15 days. Pellicle formation was regularly examined under a microscope to detect the spiral motility of Azospirillum cells. Tubes with putative azospirilla were plated on nutrient agar. Most probable numbers of Azospirillum in root macerates were 2 x 10(3)/g dry root weight in KCl, 2 x 10(4)/g dry root weight in H2O, 2 x 10(6)/g dry root weight in malate, and 3 x 10(6)/g dry root weight in the soil extract medium. Soil extract medium repressed the most abundant bacterial populations and facilitated isolation of azospirilla from a population representing less than 0.001% of the total microflora. The isolates had vibrioid to S-shaped cells with one polar flagellum when grown in liquid medium and fixed nitrogen according to the acetylene reduction assay. Their identification as Azospirillum lipoferum was based on biochemical tests and hybridization with a 16S rRNA probe specific for this species and was confirmed by the detection of a 150-MDa plasmid.